Analysis of putative biomarkers of undifferentiated spermatogonia in dog testis

Spermatogenesis begins after puberty and continues throughout a male's life, and is regulated by spermatogonial stem cells in the seminiferous tubules. Markers of male germ cells, including undifferentiated spermatogonia to fully developed spermatozoa have been identified in rodents, but not in dogs. In this study, to characterize the markers of undifferentiated spermatogonia, histological and immunohistochemical analyses were performed on pre-pubertal (1-month-old), early pubertal (4-month-old), and post-pubertal (7-month-old) dog testes. Expression of chemokine receptor 4 (CXCR4), insulin-like growth factor binding protein 3 (IGFBP3), LIN28, and Sal-like protein 4 (SALL4) genes was confirmed by immunohistochemical analysis. In pre-pubertal and early pubertal dog testes, CXCR4, IGFBP4, and LIN28 genes were expressed in undifferentiated spermatogonia, whereas the SALL4 gene was not expressed in the pre-pubertal stage. In adult dog testes, CXCR4 and IGFBP3 gene expression was detected in undifferentiated spermatogonia and co-localized with protein gene product 9.5 (PGP9.5) near the basement membrane of the seminiferous tubules. The LIN28 and SALL4 genes were expressed in synaptonemal complex protein 3-positive spermatocytes. The CXCR4 and IGFBP3 gene expression is conserved among other species, while LIN28 and SALL4 gene expression varies. Based on results of the present study, it is suggested that undifferentiated spermatogonia markers detected in other species are conserved in dogs. These results may facilitate further studies of the cellular mechanisms of spermatogenesis in dogs.